Dear all, thank you so much for all the invaluable tips that were delivered
faster than FedEx (and sorry for not being able to reply to individual
emails). My problem was resolved by adding EDTA (to about 5 mM), spinning
down at 15K for 10min to get rid of precipitation (I did have some even with
t
Could you try a quick (10-15s) halide soak (Iodine) with e.g. 1M NaI?
Tim
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Fri, 15 Feb 2008, john peter wrote:
Dear Folks,
I recently grew crystals in the presence of 100 mM Imidazole pH
Firstly - please forgive my last 'blank' message - comes of trying to
e-mail whilst travelling on a train!
I would suggest that you change the affinity tag that you're purifying
with. Go with either a GST or Strep-tag. That way you are avoiding the
imidazole that is obviously causing problems wi
Hi
There is a nice article by PSI in Nature Methods
(http://www.nature.com/nmeth/journal/v5/n2/abs/nmeth.f.202.html) about the
generic buffers for Ni column. The article or the magazine issue is a very
good reference even if you don't like those throughput guys.
One minor detail, the imidazole st
Dear Folks,
I recently grew crystals in the presence of 100 mM Imidazole pH 8.0 and 1
M Ammonium Phosphate. The protein was in 25 mM Hepes pH 7.4, 10 mM EDTA, 10
mM EGTA and 100 mM NaCl. I like to make heavy atom derivatives with Hg, Pt,
Sm, Au and Pb compounds. I think the imidazole will inter
--
Dr Antony W Oliver
Cancer Research UK: DNA Repair Enzymes Group
Section of Structural Biology
The Institute of Cancer Research
237 Fulham Road
LONDON SW3 6JB
[EMAIL PROTECTED]
020 7153 5488
--
>>> Juergen Bosch <[EMAIL PROTECTED]> 02/15/08 5:23 PM >>>
Not sure if somebody mentioned it already,
Jacob,
This is not a uncommon problem with Ni-chelation chromatography (on
Ni-NTA or Talon) with soluble and membrane proteins. In most of my
experiences, it is a salt problem (i.e., too little), but
hydrophobicity issues abound, as well.
On a pilot scale, just add any of the suggested a
Hi Jacob,
You could try keeping some inhibitor or other small molecules that could bind
to the protein in the test tube. This may help stabilizing the protein as soon
as it comes out of the column. One of my friends had to use an inhibitor
during the whole purification procedure (luckily this
Hi,
I also encounterd the same kind of problem with my protein. I noticed
that the protein eluted from Ni-NTA with 150mM imidazole or 300mM didn't
behave in the same manner; ie on gel filtration experiments, the 300mM
eluate totally came in the excluded volume while the 150mM eluate came
in t
Another much simpler (too simple?) way to approach this is that the
number of parameters is:
Ntorsions + Nbvalues + Nplacement*Nmonomers
Nmonomers is the number of monomers in the asymmetric unit and
Nplacement is the number of parameters needed to place each monomer, so
6 for the general cas
Not sure if somebody mentioned it already, but have you played with
other buffers than the recommended sodium phosphate from the manual ?
Most lazy people use Tris because there's a 1 M stock solution - but you
can try also other buffers as long as you stay in a pH range >pH6 Of course you sti
Dear all,
I am with the same problem of Jacob.
My protein is
precipitating, especially when I 'freeze it'. I am using a His Trap HP column
coupled with a desalting column. Already tried to elute with gradient of
imidazole (that allowed an elution with less amount of imidazole), but
even then, t
If You are interested in practical protein crystallization courses, have
a look at the following link:
http://www.iobcr.org/science.html
J.
--
Jeroen Raymundus Mesters, Ph.D.
Institut fuer Biochemie, Universitaet zu Luebeck
Zentrum fuer Medizinische Struktur und Zellbiologie
Ratzeburger Allee
Bernhard
Sorry I left out chiral restraints: these do not contribute to the
restraint count at all because they do not reduce the effective no of
parameters, they merely select between 2 alternative minima. As you say
the magnitude of the chiral volume (though obviously not the sign) is
completel
Jacob,
You can try several things, including the stuff already mentioned by others
- EDTA, salt, etc. A very useful option to keep in mind is to check that
excess imidazole isn't causing the problem. You can find out by simply
diluting the fractions down, or by changing the resin. His-SELECT an
first aid panic rescue might be to quickly add more salt.
(e.g. in couple of steps to 0.5 M-1 M NaCl and see if it clears up).
then add EDTA/or/and dialyse. (have to get teh salt out probably...)
the Ni-ions leaking out are a likely cause. among others.
tommi
Quoting José Trincão <[EMAIL P
To try and answer Peter & Bernhard's questions in one go: one way of
looking at counting restraints is to think of it in terms of the
reduction in the effective number of parameters that it's equivalent to.
So a bond length restraint is equivalent to reducing the 8 parameters
for 2 bonded atoms by
Hi Jacob,
try adding a bit of edta (1mM) to remove any Ni that might come off the
column. You could also add some DTT or bME to keep cysteines reduced
(careful, add only after the edta!). In my experience the gradient did
not work very weel because the protein with have a lot of impurities.
Yo
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